The invention herein relates to an improved form of cross-correlation frequency domain fluorometry and/or phosphorimetry. This process is well-known per se, being as described, for example, in Gratton U.S. Pat. No. 4,840,485, and also in a large body of technical literature on the subject. Also, instruments for performing this process are sold by I.S.S. Inc., of 309 Windsor Road, Champaign, Ill. 61820, among others.
Instruments for performing the above processes are utilized for measurement of the fluorescence decay, phosphorescence decay, anisotropy decay of fluorescence or phosphorescence, and other known uses. These instruments differ from the more common steady-state spectrofluorometers since they provide a means to record the time evolution of the deactivation of molecules or atoms after excitation with light. Typical times involved in these processes span from 1 millisecond to 1 picosecond. Such frequency domain fluorometers (which term is intended to include corresponding phosphorimeters) are also utilized for the measurement of times involved in other molecular dynamic processes such as the rotations of molecules or parts of large molecules. Also, the apparatus may be used for the resolution of excitation/emission spectra of different fluorescence or phosphorescence molecules in a mixture; for the determination of time-resolved spectra; for the resolution of kinetics decays of fluorophores in a mixture; or for the measurement of reactions occurring in the electronic excited state.
In a frequency domain fluorometer, the excitation light beam causing fluorescent emission is amplitude-modulated by a light modulator, such as a Pockels cell, or it is intrinsically modulated when the source is a mode-locked laser or synchrotron radiation source. The fluorescence emission is phase-shifted and demodulated with respect to the excitation light beam. The shift in the phase and the demodulation are both related to the lifetime of the excited electronic level of the emitting molecule or atom, providing a means to determine the modalities of the decay.
Two types of frequency domain fluorometers are commercially available at this time:
In a first type of instrument, the excitation light beam is modulated at a certain frequency F, generally in the 0.1 KHz to 300 MHz range. The phase shift and the demodulation of the fluorescence or phosphorescence are measured using the cross-correlation technique. Measurements are repeated at different modulation frequencies, usually 10 to 20 different frequencies which are logarithmically spaced in a desired frequency interval which depends on the characteristic decay time of the fluorescent or phosphorescent molecule under investigation. This type of instrument is referred to in the literature as a "serial" frequency domain fluorometer, since the various measurements at different modulation frequencies are made in a sequence of time, one after the other.
Several models of such serial fluorometers are commercially available, for example the K2 introduced by I.S.S. in 1989 and the SLM 48000, marked by SLM Instruments. Data acquisition with instruments belonging to this class usually take from one-half hour to one hour for the collection of 10 to 20 different frequencies. These instruments offer the best sensitivity, which is an important factor when working with substance having a low fluorescence quantum yield or substances in low concentration such as proteins or other biological materials. Similarly, these instruments measure in a differential way the rotational rates of molecules without the necessity of deconvolution techniques.
A second type of instrument has also been introduced to the market, as described by Mitchell U.S. Pat. No. 4,937,457 and entitled Picosecond Multiharmonic Fourier Fluorometer. This instrument si referred to as a "parallel" frequency domain fluorometer, since data are acquired simultaneously at different modulation frequencies. Usually, about 100 different modulation frequencies are acquired simultaneously. This type of instrument can potentially reduce the acquisition time by an order of magnitude, but as a disadvantage it has very low sensitivity. The advantage obtained by the reduction in data acquisition time is thus offset by the fact that the system is only capable of studying systems with a very strong fluorescent signal. When the signal is low, which is the case encountered in most applications involving biological materials, the only way to get reasonable data from this kind of instrument is to increase the data acquisition time. Therefore, in many instances the instrument does not offer any tangible advantage over a standard serial instrument.
Also, the parallel type frequency domain fluorometer is inherently more expensive, which provides further disadvantage.
Parallel frequency domain fluorometry is described in the article by B. A. Feddersen et al. entitled Digital Parallel Acquisition in Frequency Domain Fluorometry, Rev. Sci. Instrum. Vol. 60 (1989) page 2929-2936.
By this invention, a new type of cross-correlation frequency domain fluorometer and/or phosphorimeter is provided which has a significantly reduced time required for the acquisition of a good signal having a high signal to noise ratio, when compared with the standard serial-type fluorometers. However, the apparatus of this invention also retains the high sensitivity to faint signals of serial fluorometry, while providing a speed of signal acquisition which is comparable to parallel fluorometry.